Bottom Line:
P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain.The extracellular region also contains a void at the central axis, providing a second potential pathway.The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

ABSTRACTP2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

fig5: Quantitative comparisons of current modifications by MTS reagents along the lateral window. (A) Superimposed current traces of T336C recorded in response to ATP without (black) and with (blue) MTSET application (blue bar) using a 2-min interval between traces to illustrate how modification is quantified. Traces were scaled using the current amplitude 1.5 s after ATP application. Percent modification was calculated according to: modification (%) = [(b/a)−1] × 100, where b is the normalized current after a 6-s MTSET application, and a is the current at the corresponding time in the control trace. (B) Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bars indicate positions where current reduction or potentiation was observed.

Mentions:
We examined the effects of these two MTS reagents on ATP-activated currents for each of the 15 Cys mutants that retained functional activity when expressed in HEK cells. Y55C and Q56C failed to form functional channels and were not studied further. Whole cell patch clamp recordings from these mutants revealed that the application of either MTSET or MTS-TPAE has negligible effects for most Cys mutants (Figs. 4, B and C, and 5 B). Modest current reduction was observed with MTSET for the I328C mutant (Fig. 4 D, left traces), whereas small current potentiation was recorded with MTS-TPAE (Fig. 4 D, right traces), suggesting that I328 in the lateral fenestration is accessible to both MTS reagents. The rate of MTSET modification at I328C was ∼5 × 102 M−1s−1 (n = 8), slower than the rates observed for MTSET modification of most pore-lining residues within TM2. Consistent with our expectations from the zfP2X4 structure, these results demonstrate that only one residue forming the lateral fenestrations exhibits detectable reaction with MTS reagents.

fig5: Quantitative comparisons of current modifications by MTS reagents along the lateral window. (A) Superimposed current traces of T336C recorded in response to ATP without (black) and with (blue) MTSET application (blue bar) using a 2-min interval between traces to illustrate how modification is quantified. Traces were scaled using the current amplitude 1.5 s after ATP application. Percent modification was calculated according to: modification (%) = [(b/a)−1] × 100, where b is the normalized current after a 6-s MTSET application, and a is the current at the corresponding time in the control trace. (B) Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bars indicate positions where current reduction or potentiation was observed.

Mentions:
We examined the effects of these two MTS reagents on ATP-activated currents for each of the 15 Cys mutants that retained functional activity when expressed in HEK cells. Y55C and Q56C failed to form functional channels and were not studied further. Whole cell patch clamp recordings from these mutants revealed that the application of either MTSET or MTS-TPAE has negligible effects for most Cys mutants (Figs. 4, B and C, and 5 B). Modest current reduction was observed with MTSET for the I328C mutant (Fig. 4 D, left traces), whereas small current potentiation was recorded with MTS-TPAE (Fig. 4 D, right traces), suggesting that I328 in the lateral fenestration is accessible to both MTS reagents. The rate of MTSET modification at I328C was ∼5 × 102 M−1s−1 (n = 8), slower than the rates observed for MTSET modification of most pore-lining residues within TM2. Consistent with our expectations from the zfP2X4 structure, these results demonstrate that only one residue forming the lateral fenestrations exhibits detectable reaction with MTS reagents.

Bottom Line:
P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain.The extracellular region also contains a void at the central axis, providing a second potential pathway.The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

ABSTRACTP2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.